Cd6 antibody for treatment of t-cell mediated diseases or disorders

ABSTRACT

A method of treating a T-cell mediated disease in a subject by administering to the subject a therapeutically effective amount of an antibody or fragment thereof that specifically binds to CD6 is described. Humanized antibodies useful for the method are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 62/350,218, filed on Jun. 15, 2016, which is incorporated byreference herein.

GOVERNMENT FUNDING

This work was supported, at least in part, by award number NIH NS081443and AI47392 from the Department of Health and Human Services, NationalInstitutes of Health. The United States government has certain rights inthis invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 8, 2017, isnamed CCF-024421USORD_SL.txt and is 37,492 bytes in size.

BACKGROUND

CD6 is primarily expressed on T cells. Kamoun et al., J Immunol 127:987-991 (1981). Results from previous in vitro studies using differentCD6-specific monoclonal antibodies (mAbs) are contradictory, suggestingthat CD6 can either stimulate or suppress T-cell activation. Bott etal., Int Immunol 5: 783-792 (1993); Singer et al., Immunology 88:537-543 (1996). CD6 has also long been proposed as a potential targetfor therapy of autoimmune diseases, and despite the lack of clearunderstanding of CD6's function, recently a humanized mAb against CD6has been approved for treating psoriasis in India. Jayaraman, K., NatureBiotechnology 31: 1062-1063 (2013). However, historically, in the UnitedStates, the first wave of programs aimed at using CD6-targeted reagentsin treating human diseases was dropped or slowed, due in part to thelack of in vivo data to confirm the in vitro and ex vivo studies and tothe absence of CD6 gene engineered animals to test CD6-targeted reagentsin vivo. Pinto, M. & Carmo, A. M., BioDrugs: clinicalimmunotherapeutics, biopharmaceuticals and gene therapy 27: 191-202(2013). So far, there is only one report on in vivo studies of CD6 usinggenetically engineered animals (Orta-Mascaró, M. et al. J Exp Med213(8): 1387-1397 (2016)), and the potential role of CD6 in multiplesclerosis (MS) remains elusive. Recent genomic wide association studiesfrom several groups identified CD6 as a risk gene for MS (De Jager etal., Nat Genet 41: 776-782 (2009); Heap et al., Hum Mol Genet 19:122-134 (2010)), arguing a significant role of CD6 in the pathogenesisof MS.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of treating aT-cell mediated disease or disorder in a subject by administering to thesubject a therapeutically effective amount of an antibody or fragmentthereof that specifically binds to CD6. In some embodiments, the diseaseor disorder is an autoimmune disease or transplant rejection. In furtherembodiments, the T-cell mediated disease is multiple sclerosis.

A variety of antibodies or fragment thereof that specifically binds toCD6 can be used to treat the T-cell mediated disease or disorder. Insome embodiments, the antibody is a monoclonal antibody. In otherembodiments, particular if the subject is human, the antibody is ahumanized antibody. In further embodiments, the humanized antibody isselected from the group of antibodies consisting of Fab2, Fab4, andFab6, or variants thereof including only conservative sequencemodifications.

Another aspect of the present invention provides a humanized antibody orantigen-binding fragment thereof having binding specificity for CD6,wherein the antibody selected from the group of antibodies consisting ofFab2, Fab4, and Fab6, or variants thereof including only conservativesequence modifications. In some embodiments, the humanized antibodycomprises a heavy chain comprising SEQ ID NO: 1, a light chaincomprising SEQ ID NO: 2, or variants thereof including only conservativesequence modifications. In other embodiments, the humanized antibodycomprises a heavy chain comprising SEQ ID NO: 3, a light chaincomprising SEQ ID NO: 4, or variants thereof including only conservativesequence modifications. In further embodiments, the humanized antibodycomprises a heavy chain comprising SEQ ID NO: 5, a light chaincomprising SEQ ID NO: 6, or variants thereof including only conservativesequence modifications.

Another aspect of the invention provides a kit comprising a humanizedantibody or fragment thereof that specifically binds to CD6, and apackage for holding the antibody. In some embodiments, the kit includesinstructions for using the kit to carry out a method of treating aT-cell mediated disease or disorder in a subject by administering to thesubject a therapeutically effective amount of the humanized antibody orfragment thereof that specifically binds to CD6. In additionalembodiments, the disease is an autoimmune disease such as multiplesclerosis. In yet further embodiments, the antibody is selected from thegroup of antibodies consisting of Fab2, Fab4, and Fab6, or variantsthereof including only conservative sequence modifications.

BRIEF DESCRIPTION OF THE FIGURES

The present invention may be more readily understood by reference to thefollowing figures, wherein:

FIGS. 1A-1C provide graphs and images showing the development of the CD6KO mice. A. Exons 5-7 of the CD6-coding region were replaced by aneomycin gene cassette after homologous recombination. Southern blotconfirmed the recombination event. B. CD6 KO mice genotyping. All mCD6KO mice were genotyped with a dual PCR. The neo insert can be identifiedby a PCR with a neo primer (forward-5′-CTT GGG TGG AGA GGC TAT TC-3′)(SEQ ID NO: 7) and a exon 8 (reverse-5′-AGC CAA CCT TTC TTC TGA GAGCCA-3′) (SEQ ID NO: 8). Mice heterozygous for the neo insert will alsocontain mouse CD6 exon 5 (forward-5′-TGG GCC CAA AGC ATT TAG CTT GAC-3′)(SEQ ID NO: 9) and (reverse-5′-TAC AGA GAG CTT GGC AGT GCT TGA-3′) (SEQID NO: 10). A mouse containing neo between exons 5-7 (lane 1) but alsohaving exon 5 (lane 5) is a heterozygous KO. In contrast, a homologousKO has neo (lane 2) but does not contain exon 5 (lane 6). Lane 3 is the100 base pair ladder and lane 4 is a PCR negative control. C. Absence ofCD6 protein on CD6 KO mouse lymphocytes. Lymphocytes from WT,heterozygous (Het) KO and homologous (homo) KO mice were analyzed forCD6 expression by flow cytometry, showing reduced levels of CD6 in hetKO mice (middle dotted line), and absence of CD6 in homo KO mice (leftsolid line). Neg, negative control.

FIGS. 2A-2E provide graphs and images showing that CD6 KO mice areprotected from EAE. A. Clinical scores of the WT (circles) and CD6 KOmice (squares). Combined results from four experiments. n=15 in eachgroup. *p<0.01; B. MOG-specific Th1 and Th17 responses were reduced inCD6 KO mice (*p<0.05). Splenocytes from WT and CD6 KO mice 21 days afterimmunization were incubated without peptide (white bars), with 10 μg/mLof nonrelevant peptide (IRBP₁₋₂₀, gray bars) or with 10 μg/mL MOG₇₉₋₉₆peptide (black bars) for 72 hours. IFN-γ and IL-17 levels in the culturesupernatants were measured by ELISA (n=14 in each group, *p<0.05). C.Spinal cord in CD6 KO mice had markedly reduced leukocyte infiltrationas assessed by H&E staining D. Representative images of spinal cordsections from the EAE mice after H&E staining, showing that CD6 KO micehave significantly reduced cell infiltration. (Magnification: Upper, 4×;Insets shown at Lower, 20×). E. Representative images of spinal cordsections from the EAE mice after Luxol Blue staining, showing that CD6KO mice had intact myelin sheath (blue staining) compared with severedemyelination in the WT mice. (Magnification: 4×).

FIGS. 3A-3D provide graphs and images showing that absence of CD6 leadsto impaired Th1 and Th17 development. Purified CD4⁺T cells from naïve WTand CD6 KO mice were activated and cultured under Th1 or Th17polarization conditions for 5 days. Then the development of Th1 and Th17was assessed by intracellular staining of IFN-γ or IL-17a. A and B.Representative results of the intracellular IFN-γ or IL-17a stainingwithin the differentiated T cells. C and D. Combined results from threedifferent experiments. n=3, data are mean±SEM, *p<0.05.

FIGS. 4A-4B provide graphs showing that absence of CD6 leads toaugmented T cell activation. Purified CD4⁺T cells from naïve WT and CD6KO mice were activated by incubation with 1 μg/mL anti-CD3 and anti-CD28mAbs, then the activation of cells was assessed 5 hours later bymeasuring the up-regulation of activation markers CD25 (A) and CD69 (B)using flow cytometric analysis. Gray bars, before activation; blackbars, 5 hours after activation, n=5, data are mean±SEM, *p<0.05.

FIGS. 5A-5D provide graphs showing that absence of CD6 leads to reducedproliferation and enhanced apoptosis of activated T cells. PurifiedCD4⁺T cells from naïve WT and CD6 KO mice were activated by incubationwith plate-bound anti-CD3 and anti-CD28 mAbs, then cultured under theTh1 or Th17 polarization conditions. At 5, 24, 48, and 72 hour timepoints, proliferation of the activated cells was assessed by measuringBrdU incorporation using a BrdU ELISA kit (A and B), and apoptosis ofthe activated T cells was assessed by staining the cells with annexin Vfollowed by flow cytometric analysis (C and D). n=3 in each group, dataare mean±SEM, *p<0.05.

FIGS. 6A-6C provide graphs and images showing the absence of CD6 reducesT-cell migration through BMEC monolayers. WT DBA-1 mouse BMECs werefirst isolated by following an established protocol (A) and were ˜90%pure based on CD34 staining (B). The isolated BMECs were grown onculture inserts until monolayers were formed, then 0.6×10⁶ ofCFSE-labeled and anti-CD3/CD28 mAb-activated T cells from naïve WT andCD6 KO mice were added into the culture inserts with 20 ng/mL culture,cells that remained in the culture inserts and those that migrated intothe lower chambers were quantitated (C). n=4 in each group, data aremean±SEM, *pp<0.05.

FIGS. 7A-7G provide graphs and images showing that mouse anti-human CD6mAb (UMCD6) treats EAE in CD6 humanized mice. A and B. CD6 humanizedmice do not express mouse CD6 (A) but express human CD6 (B). C. EAEclinical scores of CD6 humanized mice treated with UMCD6 and controlmouse IgGs. CD6 humanized mice were immunized to induced EAE. After miceshowed mild clinical symptoms they were randomly divided into two groupswith one group receiving ˜0.4 mg per mouse anti-human CD6 IgG (UMCD6)(circles) and the other receiving 0.4 mg purified mouse IgGs (squares).Clinical scores were recorded daily. n=14 in each group, *p<0.05. D andE. Th1 (D) and Th17 (E) recall assays in CD6 humanized mice treated withUMCD6 or control IgG. At the end of treatment experiments (day 14),splenocytes from mice were collected and incubated without peptide(light gray bars), with 10 μg/mL of a nonrelevant peptide (IRBP₁₋₂₀,dark gray bars) or with 10 μg/mL MOG₇₉₋₉₆ peptide (black bars) for 72hours. IFN-γ and IL-17a levels in the culture supernatants were measuredby ELISA. n=7 in each group, *p<0.05. F and G. Representative images ofspinal cord sections showing significantly reduced cell infiltration (F)and demyelination (G) in the UMCD6-treated mice compared with thecontrols. (Magnification: 4×).

FIGS. 8A-8B provide graphs and images showing that UMCD6 treatment doesnot deplete T cells. CD4⁺ and CD8⁺T cell percentages in the peripheralblood were analyzed in both UMCD6 and control IgG-treated CD6 humanizedmice by flow cytometry at the end of the treatment studies, showing nodifference between the two groups of mice. n=14 in each group. A.FSC/SSC shows the lymphocyte population analyzed. B. CD4⁺ and CD8⁺T cellpercentages of mice treated with UMCD6 (black bars) or control IgG (graybars).

FIG. 9 provides a sensorgram showing the off-rate analysis of secretedFab antibodies to rhCD6-Fc. The top 6 high affinity binders wereselected for single cycle kinetics analysis.

FIG. 10 provides a sensorgram of a single cycle kinetics analysis ofsecreted Fab antibodies to rhCD6-Fc. Only the affinity of Fab2 iscomparable to WT Fab (parent chimeric antibody).

FIG. 11 provides graphs showing cell binding validation of 11 selectedhumanized clones by FACS using Jurkat cells.

FIG. 12 provides graphs showing cell binding validation of 6 selectedFab clones by FACS using Jurkat cells.

FIG. 13 provides SDS-PAGE analysis of three purified humanizedantibodies. Lane 1, 2 μg purified Humanized IgG2 under non-reducingcondition; Lane 2, 2 μg purified Humanized IgG4 under non-reducingcondition; Lane 3, 2 μg purified Humanized IgG6 under non-reducingcondition; Lane 4, 2 μg purified Humanized IgG2 under reducingcondition; Lane 5, 2 μg purified Humanized IgG4 under reducingcondition; Lane 6, 2 μg purified Humanized IgG6 under reducingcondition; Lane M, page ruler pre-stained protein ladder (ThermoScientific, Cat. No.: 26616);

FIG. 14 provides graphs showing the fitting of BIAcore experimental datato a 1:1 binding model.

FIG. 15 provides graphs showing the temperature-induced denaturation ofparent chimeric antibody and humanized antibodies monitored by thechanges in ellipticity at 202 and 208 nm. Smooth curves are the fittingof CD experimental data to a two-state model.

FIGS. 16A and 16B provide the (A) DNA (SEQ ID NO: 18) and (B) amino acidsequences (SEQ ID NO: 19) for the V_(H)2-hIgG1C_(H) antibody fragment.

FIGS. 17A and 17B provide the (A) DNA (SEQ ID NO: 20) and (B) amino acidsequences (SEQ ID NO: 21) for the V_(H)4-hIgG1C_(H) antibody fragment.

FIGS. 18A and 18B provide the (A) DNA (SEQ ID NO: 22) and (B) amino acidsequences (SEQ ID NO: 23) for the V_(H)4-hIgG1C_(H) antibody fragment.

FIGS. 19A and 19B provide the (A) DNA (SEQ ID NO: 24) and (B) amino acidsequences (SEQ ID NO: 25) for the V_(L)-hIgKC_(L) antibody fragment.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which these exemplary embodiments belong. The terminologyused in the description herein is for describing particular exemplaryembodiments only and is not intended to be limiting of the exemplaryembodiments. As used in the specification and the appended claims, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present embodiments. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldbe construed in light of the number of significant digits and ordinaryrounding approaches.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

“Treating”, as used herein, means ameliorating the effects of, ordelaying, halting or reversing the progress of a disease or disorder.The word encompasses reducing the severity of a symptom of a disease ordisorder and/or the frequency of a symptom of a disease or disorder.

A “subject”, as used therein, can be a human or non-human animal.Non-human animals include, for example, livestock and pets, such asovine, bovine, porcine, canine, feline and murine mammals, as well asreptiles, birds and fish. Preferably, the subject is human.

The language “effective amount” or “therapeutically effective amount”refers to a nontoxic but sufficient amount of the composition used inthe practice of the invention that is effective to provide effectivetreatment in a subject. That result can be reduction and/or alleviationof the signs, symptoms, or causes of a disease or disorder, or any otherdesired alteration of a biological system. An appropriate therapeuticamount in any individual case may be determined by one of ordinary skillin the art using routine experimentation.

The term antibody, as used herein and unless further limited, refers tosingle chain, two-chain, and multi-chain proteins and glycoproteinsbelonging to the classes of polyclonal, monoclonal, chimeric and heteroimmunoglobulins; it also includes synthetic and genetically engineeredvariants of these immunoglobulins. The term “Antibody fragment” includesFab, Fab′, F(ab′)2, and Fv fragments, as well as any portion of anantibody having specificity toward a desired target epitope or epitopes.

The term monoclonal antibody, as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variants that mayarise during production of the monoclonal antibody, such variantsgenerally being present in minor amounts. In contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different epitopes, each monoclonal antibody isdirected against a single epitope on the antigen. In addition to theirspecificity, the monoclonal antibodies are advantageous in that they areuncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies.

The term chimeric antibody, as used herein, refers to an antibody whichincludes sequences derived from two different antibodies, whichtypically are of different species. Most typically, chimeric antibodiesinclude human and non-human antibody fragments, generally human constantand non-human variable regions.

The term humanized antibody, as used herein, refers to a type ofchimeric antibody derived from a non-human antibody, and a humanantibody which retains or substantially retains the antigen-bindingproperties of the parent antibody but which is less immunogenic inhumans.

The term antigen, as used herein, refers to a molecule or a portion of amolecule capable of being bound by an antibody which is additionallycapable of inducing an animal to produce an antibody capable of bindingto an epitope of that antigen. An antigen can have one or more than oneepitope. The specific reaction referred to above is meant to indicatethat the antigen will react, in a highly selective manner, with itscorresponding antibody and not with the multitude of other antibodieswhich can be evoked by other antigens.

The term epitope, as used herein, refers to that portion of any moleculecapable of being recognized by, and bound by, an antibody. In general,epitopes consist of chemically active surface groupings of molecules,for example, amino acids or sugar side chains, and have specificthree-dimensional structural characteristics as well as specific chargecharacteristics. The epitopes of interest for the present invention areepitopes comprising amino acids.

As used herein, a humanized antibody comprises heavy or light chainvariable framework regions that are “the product of” or “derived from” aparticular human germline sequence (human gene) if the variableframework regions of the antibody are obtained from a system that useshuman germline immunoglobulin genes. Such systems include immunizing atransgenic mouse carrying human immunoglobulin genes with the antigen ofinterest or screening a human immunoglobulin gene library displayed onphage with the antigen of interest. A humanized antibody which comprisesa heavy or light chain variable framework region that is “the productof” or “derived from” a human germline immunoglobulin sequence can beidentified as such by comparing the amino acid sequence of the heavy orlight chain variable framework region of the humanized antibody to theamino acid sequences of the heavy or light chain variable frameworkregion of human germline immunoglobulins. A humanized antibody thatcomprises a heavy or light chain variable framework region that is “theproduct of” a particular human germline immunoglobulin sequence has aheavy or light chain variable framework region which is 100% identicalin amino acid sequence to the heavy or light chain variable frameworkregion of the particular human germline immunoglobulin sequence. Ahumanized antibody that comprises a heavy or light chain variableframework region that is “derived from” a particular human germlineimmunoglobulin sequence may contain amino acid differences as comparedto the heavy or light chain variable framework region of the particulargermline sequence, due to, for example, naturally-occurring somaticmutations or intentional introduction of site-directed mutation.However, a selected humanized antibody typically is at least 90%identical in amino acid sequence of the heavy or light chain variableframework region to an amino acid sequence encoded by the heavy or lightchain variable framework region of a human germline immunoglobulin geneand contains amino acid residues that identify the humanized antibody asbeing derived from human when compared to the germline immunoglobulinamino acid sequences of other species (e.g., murine germline sequences).In certain cases, a humanized antibody may be preferably at least 95%,more preferably at least 96%, most preferably at least 97%, inparticular at least 98%, most particular at least 99%, identical inamino acid sequence of the heavy or light chain variable frameworkregion to the amino acid sequence of the heavy or light chain variableframework region encoded by the germline immunoglobulin gene. Typically,the heavy or light chain variable framework region of a humanizedantibody derived from a particular human germline sequence will displayno more than 10 amino acid, preferably no more than 5, or even morepreferably no more than 4, 3, 2, or 1 differences from the amino acidsequence of the heavy or light chain variable framework region encodedby the human germline immunoglobulin gene.

The term “Fab” or “Fab region” as used herein includes the polypeptidesthat comprise the VH, CHL VL, and CL immunoglobulin domains. Fab mayrefer to this region in isolation, or this region in the context of afull length antibody or antibody fragment.

The term “Fc” or “Fc region”, as used herein includes the polypeptidecomprising the constant region of an antibody excluding the firstconstant region immunoglobulin domain. Thus Fc refers to the last twoconstant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains.

The term “variant antibody” or “antibody variant” as used hereinincludes an antibody sequence that differs from that of a parentantibody sequence by virtue of at least one amino acid modificationcompared to the parent. The variant antibody sequence herein willpreferably possess at least about 80%, most preferably at least about90%, more preferably at least about 95% amino acid sequence identitywith a parent antibody sequence. Antibody variant may refer to theantibody itself, compositions comprising the antibody variant, or theamino acid sequence that encodes it.

Antibody fragments include, but are not limited to, (i) the Fab fragmentconsisting of VL, VH, CL and CH1 domains, including Fab′ and Fab′-SH,(ii) the Fd fragment consisting of the VH and CH1 domains, (iii) the Fvfragment consisting of the VL and VH domains of a single antibody; (iv)the dAb fragment (Ward et al., Nature 341:544-546 (1989)) which consistsof a single variable, (v) F(ab′)2 fragments, a bivalent fragmentcomprising two linked Fab fragments (vi) single chain Fv molecules(scFv), wherein a VH domain and a VL domain are linked by a peptidelinker which allows the two domains to associate to form an antigenbinding site (Bird et al., Science 242:423-426 (1988); Huston et al.,Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883 (1988)), (vii) bispecificsingle chain Fv dimers (PCT/US92/09965), (viii) “diabodies” or“triabodies”, multivalent or multi-specific fragments constructed bygene fusion (Tomlinson et. al., Methods Enzymol. 326:461-479 (2000);WO94/13804; Holliger et al., Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448(1993)) and (ix) scFv genetically fused to the same or a differentantibody (Coloma & Morrison, Nature Biotechnology 15, 159-163 (1997)).

Antibodies are grouped into classes, also referred to as isotypes, asdetermined genetically by the constant region. Human constant lightchains are classified as kappa (Cκ) and lambda (Cλ) light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. TheIgG class is the most commonly used for therapeutic purposes. In humansthis class comprises subclasses IgG1, IgG2, IgG3, and IgG4. In mice thisclass comprises subclasses IgG1, IgG2a, IgG2b, IgG3. IgM has subclasses,including, but not limited to, IgM1 and IgM2. IgA has severalsubclasses, including but not limited to IgA1 and IgA2. Thus, “isotype”as used herein is meant any of the classes or subclasses ofimmunoglobulins defined by the chemical and antigenic characteristics oftheir constant regions. The known human immunoglobulin isotypes areIgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE.

As used herein, an antibody “specifically binds”, referring to anantibody binding to a target structure, means that the antibody binds atarget structure, or subunit thereof, but does not bind to a biologicalmolecule that is not a target structure. Antibodies that specificallybind to a target structure, or subunit thereof, do not cross-react withbiological molecules that are outside the target structure family. Anantibody specific for CD6 can be an antibody or antibody fragmentcapable of binding to that specific protein with a specific affinity ofbetween 10⁻⁸ M and 10⁻¹¹ M. In some embodiments, an antibody or antibodyfragment binds to a selected antigen with a specific affinity of greaterthan 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, or 10⁻¹¹ M, between 10⁻⁸ M-10⁻¹¹M, 10⁻⁹ M-10⁻¹⁰ M, and 10⁻¹⁰ M-10⁻¹¹ M. In a preferred aspect, specificactivity is measured using a competitive binding assay as set forth inAusubel FM, (1994). Current Protocols in Molecular Biology. Chichester:John Wiley and Sons (“Ausubel”), which is incorporated herein byreference.

The term “amino acid modification” herein includes an amino acidsubstitution, insertion, and/or deletion in a polypeptide sequence. By“amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with another amino acid. For example, thesubstitution R94K refers to a variant polypeptide, in this case a heavychain variable framework region variant, in which the arginine atposition 94 is replaced with a lysine. For the preceding example, 94Kindicates the substitution of position 94 with a lysine. For thepurposes herein, multiple substitutions are typically separated by aslash. For example, R94K/L78V refers to a double variant comprising thesubstitutions R94K and L78V. By “amino acid insertion” or “insertion” asused herein is meant the addition of an amino acid at a particularposition in a parent polypeptide sequence. For example, insert −94designates an insertion at position 94. By “amino acid deletion” or“deletion” as used herein is meant the removal of an amino acid at aparticular position in a parent polypeptide sequence. For example, R94−designates the deletion of arginine at position 94.

As used herein, the term “conservative modifications” or “conservativesequence modifications” is intended to refer to amino acid modificationsthat do not significantly affect or alter the binding characteristics ofthe antibody containing the amino acid sequence. Such conservativemodifications include amino acid substitutions, insertions anddeletions. Modifications can be introduced into an antibody of theinvention by standard techniques known in the art, such as site-directedmutagenesis and PCR-mediated mutagenesis. Conservative amino acidsubstitutions are ones in which the amino acid residue is replaced withan amino acid residue having a similar side chain. Families of aminoacid residues having similar side chains have been defined in the art.These families include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, one or more amino acidresidues within the constant or variable regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family and the altered antibody (variant antibody) can betested for retained function.

Methods of Treating T-Cell Mediated Disease

In one aspect, the present invention provides a method of treating aT-cell mediated disease or disorder in a subject by administering to thesubject a therapeutically effective amount of an antibody or fragmentthereof that specifically binds to CD6.

In some embodiments of the invention, the T-cell mediated disease ordisorder is an autoimmune disease. Examples of T-cell mediatedautoimmune diseases include multiple sclerosis, polymyositis, acutedisseminated encephalomyelitis, Balo's disease, clinically isolatedsyndrome, HTLV-I associated myelopathy, neuromyelitis optica, Schilder'sdisease, and transverse myelitis. In other embodiments, the T-cellmediated disease or disorder is transplantation rejection. In transplantrejection, transplanted tissue is rejected by the recipient's immunesystem, which damages or destroys the transplanted tissue. Alloreactivekiller T cells, also called cytotoxic T lymphocytes (CTLs), have CD8receptors that dock to the transplanted tissue's MHC class I molecules,which display the donor's self peptides, and trigger the target cell'sprogrammed cell death by apoptosis. T-cell mediated transplant rejectionis typically acute rejection.

In some embodiments, the T-cell mediated disease or disorder is multiplesclerosis. Multiple sclerosis is an inflammatory disease in which theinsulating covers of nerve cells in the brain and spinal cord aredamaged. This damage disrupts the ability of parts of the nervous systemto communicate. Specific symptoms of multiple sclerosis are determinedby the locations of the lesions within the nervous system, and mayinclude loss of sensitivity or changes in sensation such as tingling,pins and needles or numbness, muscle weakness, very pronounced reflexes,muscle spasms, or difficulty in moving; difficulties with coordinationand balance (ataxia); problems with speech or swallowing, visualproblems (nystagmus, optic neuritis or double vision), feeling tired,acute or chronic pain, and bladder and bowel difficulties, among others.

The antibody (e.g., humanized immunoglobulin) is administered in aneffective amount which inhibits binding of CD6 to a ligand thereof. Fortherapy, an effective amount will be sufficient to achieve the desiredtherapeutic (including prophylactic) effect (such as an amountsufficient to reduce or prevent CD6-mediated binding and/or signalling).The antibody can be administered in a single dose or multiple doses. Thedosage can be determined by methods known in the art and can bedependent, for example, upon the individual's age, sensitivity,tolerance and overall well-being. Suitable dosages for antibodies can befrom about 0.1 mg/kg body weight to about 10.0 mg/kg body weight pertreatment.

According to the method, the antibody (e.g., humanized immunoglobulin)can be administered to an individual (e.g., a human) alone or inconjunction with another agent. A humanized immunoglobulin can beadministered before, along with or subsequent to administration of theadditional agent. Thus, the invention includes pharmaceuticalcompositions comprising an anti-CD6 antibody or fragment thereof of theinvention and a suitable carrier. In one embodiment, more than oneanti-CD6 antibody which inhibits the binding of CD6 to its ligand(s) isadministered. In another embodiment, an additional pharmacologicallyactive ingredient (e.g., an agent suitable for treating multiplesclerosis, such as an interferon, fingolimod, teriflunomide, dimethylfumarate, glatiramer acetate, methotrexate, or natalizumab) can beadministered in conjunction with an anti-CD6 antibody of the presentinvention. A variety of routes of administration are possible,including, but not necessarily limited to, parenteral (e.g.,intravenous, intraarterial, intramuscular, subcutaneous injection), oral(e.g., dietary), topical, inhalation (e.g., intrabronchial, intranasalor oral inhalation, intranasal drops), or rectal, depending on thedisease or condition to be treated. Parenteral administration is apreferred mode of administration.

Formulation will vary according to the route of administration selected(e.g., solution, emulsion). An appropriate composition comprising theanti-CD6 antibody to be administered can be prepared in aphysiologically acceptable vehicle or carrier. For solutions oremulsions, suitable carriers include, for example, aqueous oralcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles can include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles can include variousadditives, preservatives, or fluid, nutrient or electrolyte replenishers(See, generally, Remington's Pharmaceutical Sciences, 17th Edition, MackPublishing Co., PA, 1985). For inhalation, the compound can besolubilized and loaded into a suitable dispenser for administration(e.g., an atomizer, nebulizer or pressurized aerosol dispenser).

The antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to the target antigen in the patient.In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μg/ml and in some methods about 25-300μg/ml. Alternatively, antibody can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of theantibody in the patient. The dosage and frequency of administration canvary depending on whether the treatment is prophylactic or therapeutic.In prophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated.

The selected dosage level will depend upon a variety of pharmacokineticfactors including the activity of the particular compositions of thepresent invention employed, the route of administration, the time ofadministration, the rate of excretion of the particular antibody beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

CD6 Antibodies

The present invention involves administering a therapeutically effectiveamount of an antibody or fragment thereof that specifically binds to CD6to a subject. CD6 (Cluster of Differentiation 6) is a human proteinencoded by the CD6 gene, found on the outer membrane of T-lymphocytes aswell as some other immune cells. The encoded protein contains threescavenger receptor cysteine-rich (SRCR) domains and a binding site foran activated leukocyte cell adhesion molecule. In some embodiments, theantibody is a monoclonal antibody. Preparation of immunizing antigen,and polyclonal and monoclonal antibody production can be performed asdescribed herein, or using other suitable techniques. A variety ofmethods have been described (see e.g., Kohler et al., Nature, 256:495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al.,Nature 266: 550-552 (1977); Koprowski et al., U.S. Pat. No. 4,172,124;Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (ColdSpring Harbor Laboratory: Cold Spring Harbor, N.Y.); Current ProtocolsIn Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M.et al., Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, (1991)).Generally, a hybridoma can be produced by fusing a suitable immortalcell line (e.g., a myeloma cell line such as SP2/0) with antibodyproducing cells. The antibody producing cell, preferably those of thespleen or lymph nodes, are obtained from animals immunized with theantigen of interest. The fused cells (hybridomas) can be isolated usingselective culture conditions, and cloned by limiting dilution. Cellswhich produce antibodies with the desired binding properties can beselected by a suitable assay (e.g., ELISA).

In some embodiments, the anti-CD6 antibody is a humanized antibody.Humanized antibodies are preferable for use in human subjects, in orderto avoid generating an immune response against the antibodiesthemselves. Examples of humanized antibodies include those selected fromthe group of antibodies consisting of Fab2, Fab4, and Fab6, or variantsthereof including only conservative sequence modifications. Otherexamples of humanized antibodies include those prepared by theinventors, as described herein.

In some embodiments, the humanized antibody comprises a heavy chaincomprising the amino acid sequenceQVQLQESGPGLVKPSETLSLTCTVSGFSLSRYSVHWVRQPPGKGLEWLGLIWGGGFTDYNSALKSRLTISKDNSKNQVSLKLSSVTAADTAVYYCAREGVAYWGQGTLVTVSS (SEQ ID NO: 1),a light chain comprising the amino acid sequenceDVVMTQSPLSLPVTLGQPASISCKSSQSLLNSDGRTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGPGTKVDIK (SEQ ID NO: 2),or variants thereof including only conservative sequence modifications.

In some embodiments, the humanized antibody comprises a heavy chaincomprising the amino acid sequenceQVQLQESGPGLVKPSETLSLTCTVSGFSISRYSVHWIRQPPGKGLEWIGLIWGGGFTDYNSALKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAREGVAYWGQGTLVTVSS (SEQ ID NO: 3),a light chain comprising the amino acid sequenceDVVMTQSPLSLPVTLGQPASISCKSSQSLLNSDGRTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGPGTKVDIK (SEQ ID NO: 4),or variants thereof including only conservative sequence modifications.

In some embodiments, the humanized antibody comprises a heavy chaincomprising the amino acid sequenceQVQLQESGPGLVKPSETLSLTCTVSGFSLSRYSVHWIRQPPGKGLEWIGLIWGGGFTDYNSALKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAREGVAYWGQGTLVTVSS (SEQ ID NO: 5),a light chain comprising the amino acid sequenceDVVMTQSPLSLPVTLGQPASISCKSSQSLLNSDGRTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGPGTKVDIK (SEQ ID NO: 6),or variants thereof including only conservative sequence modifications.

Humanized immunoglobulins can be produced using synthetic and/orrecombinant nucleic acids to prepare genes (e.g., cDNA) encoding thedesired humanized chain. For example, nucleic acid (e.g., DNA) sequencescoding for humanized variable regions can be constructed using PCRmutagenesis methods to alter DNA sequences encoding a human or humanizedchain, such as a DNA template from a previously humanized variableregion (see e.g., Kamman, M., et al., Nucl. Acids Res., 17: 5404(1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993);Daugherty, B. L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991);and Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302 (1991)). Usingthese or other suitable methods, variants can also be readily produced.In one embodiment, cloned variable regions can be mutagenized, andsequences encoding variants with the desired specificity can be selected(e.g., from a phage library; see e.g., Krebber et al., U.S. Pat. No.5,514,548; Hoogenboom et al., WO 93/06213).

The antigen binding region of the humanized immunoglobulin (the nonhumanportion) can be derived from an immunoglobulin of nonhuman origin(referred to as a donor immunoglobulin) having binding specificity forCD6. For example, a suitable antigen binding region can be derived froma murine monoclonal antibody having binding specificity for CD6. Othersources include CD6-specific antibodies obtained from nonhuman sources,such as rodent (e.g., mouse, rat), rabbit, pig goat or non-human primate(e.g., monkey). Additionally, other polyclonal or monoclonal antibodies,such as antibodies which bind to the same or similar epitope as the 1D9antibody, can be made (e.g., Kohler et al., Nature, 256:495-497 (1975);Harlow et al., 1988, Antibodies: A Laboratory Manual, (Cold SpringHarbor, N.Y.); and Current Protocols in Molecular Biology, Vol. 2(Supplement 27, Summer '94), Ausubel et al., Eds. (John Wiley & Sons:New York, N.Y.), Chapter 11 (1991)).

For example, antibodies can be raised against an appropriate immunogenin a suitable mammal (e.g., a mouse, rat, rabbit or sheep). Cellsbearing CD6, membrane fractions containing CD6, and immunogenicfragments of CD6 are examples of suitable immunogens. Antibody-producingcells (e.g., a lymphocyte) can be isolated from, for example, the lymphnodes or spleen of an immunized animal. The cells can then be fused to asuitable immortalized cell (e.g., a myeloma cell line), thereby forminga hybridoma. Fused cells can be isolated employing selective culturingtechniques. Cells which produce antibodies with the desired specificitycan be selected by a suitable assay (e.g., ELISA) Immunoglobulins ofnonhuman origin having binding specificity for CD6 can also be obtainedfrom antibody libraries (e.g., a phage library comprising nonhuman Fabmolecules).

Kits

Another aspect of the invention provides a kit comprising a humanizedantibody or fragment thereof that specifically binds to CD6, and apackage for holding the antibody. Suitable packages include, forexample, bottles, vials or syringes. The package may be formed from avariety of materials such as glass or plastic. The container holds acomposition that may be effective for treating the condition and mayhave a sterile access port (e.g., the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition may bethe anti-CD6 antibody described herein. The label or package insert mayindicate that the composition may be used for treating the condition ofchoice, such as cancer. In one embodiment, the label or package insertmay indicate that the composition comprising the anti-CD6 antibody maybe used to treat a T-cell mediated disease or disorder.

The reagents may be supplied in a solid (e.g., lyophilized) or liquidform. The kits of the present invention may optionally comprisedifferent containers (e.g., vial, ampoule, test tube, flask or bottle)for each individual buffer, cell type and/or reagent. Each componentwill generally be suitable as aliquoted in its respective container orprovided in a concentrated form. Other containers suitable forconducting certain steps of the disclosed methods may also be provided.The individual containers of the kit are preferably maintained in closeconfinement for commercial sale. For example, the kit can include acarrier for the various components of the kit. The carrier can be acontainer or support in the form of, e.g., a bag, box, tube or rack, andis optionally compartmentalized. The carrier may define an enclosedcontainer for safety purposes during shipment and storage.

The antibody or fragment thereof that specifically binds to CD6 can beany of the antibodies described herein. For example, in some embodimentsthe antibody is selected from the group of antibodies consisting ofFab2, Fab4, and Fab6, or variants thereof including only conservativesequence modifications.

In some embodiments, the kit further includes instructions for using thekit to carry out a method of treating a T-cell mediated disease ordisorder in a subject by administering to the subject a therapeuticallyeffective amount of the humanized antibody or fragment thereof thatspecifically binds to CD6. For example, in some embodiments, the kitincludes instructions for carrying out a method of treating multiplesclerosis. While the instructions are typically written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this disclosure. Such media include, but are not limited to,electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. As used herein, theterm “instructions” can include the address of an internet site thatprovides the instructions.

Examples have been included to more clearly describe a particularembodiment of the invention and its associated cost and operationaladvantages. However, there are a wide variety of other embodimentswithin the scope of the present invention, which should not be limitedto the particular examples provided herein.

EXAMPLES Example 1: CD6 as a Target for Treatment of a Model of MultipleSclerosis

CD6 has been recently identified as a risk gene for multiple sclerosis(MS), an autoimmune disease in which myelin-reactive T cells playimportant roles in pathogenesis. However, partially due to the lack ofan animal model, the role of CD6 on regulating T cells is contradictorybased on in vitro studies, and its potential as a new target for MStreatment remains unclear. Here, the generation of a CD6 knockout mouseand a CD6 humanized mouse is reported. It is demonstrated that (a) CD6knockout mice are protected from central nervous system injuries inexperimental autoimmune encephalomyelitis (EAE), an animal model of MS,(b) CD6 is a negative regulator of T cell activation, but a positiveregulator of T cell proliferation and survival. Therefore, lack of CD6leads to reduced T-cell responses in EAE; (c) CD6 on T cells is alsorequired for T-cell infiltration through the blood-brain barrier (BBB)into the central nervous system (CNS), and (d) systemic administrationof an anti-human CD6 antibody after disease onset reverses EAEprogression in CD6 humanized mice. These data demonstrate that targetingCD6 should be effective in treating MS.

Methods

Development of the CD6 Knockout (KO) Mice:

CD6 KO mice were developed following a conventional KO mice generationprotocol. In this mouse, multiple exons including exons 5-7 of the CD6gene were replaced by a neomycin cassette after homologousrecombination, resulting in the absence of CD6 protein. The resultantCD6 KO mice were backcrossed with DBA-1 mice for 12 generations. Asanticipated, flow cytometry showed that lymphocytes from the CD6knockout mice are deficient in CD6 protein and these mice have the samenumbers of CD4⁺ and CD8⁺T cells compared with WT DBA-1 mice (FIG. 1).

Induction and Assessment of Disease Severity of EAE:

EAE was induced by active immunization, and disease severity wasassessed by assigning clinical scores following previously publishedprotocols. Li et al., Mol Immunol 46: 2885-2891 (2009). In brief, 8-10weeks old female mice were immunized at base of tail and both thighswith 200 μg of mouse MOG₇₉₋₉₆ peptide (custom synthesized by Genscript,NJ) emulsified in Complete Freund's Adjuvant (CFA) (Sigma, MO) that hadbeen supplemented with M. tuberculosis strain H37RA to 4 mg/ml. 0.2 μgof Pertussis toxin (List Biological Laboratories, CA) was injected i.p.right after immunization and the following day. Clinical severity wasassessed daily with a 0 to 5 scoring system (0, no signs; 1, flaccidtail; 2, impaired righting reflex and/or gait; 3, partial hind limbparalysis; 4, total hind limb paralysis; 5, moribund or dead). Eachmouse was assessed twice within the same day, and the average score fromthe two assessments was recorded as the score for that day.

Histology and Histochemical Staining of Spinal Cords:

At time of sacrifice, spinal cords were removed and fixed in 10%(vol/vol) formalin in PBS buffer for 24 hours, then embedded inparaffin. Sections were cut at 5 μm on a microtome and stained byhaematoxylin and eosin (H&E) to assess CNS inflammatory infiltrates andLuxol Fast Blue (LFB) to assess demyelinated areas following standardprotocols.

Th1 and Th17 Recall Assays:

At time of sacrifice, splenocytes were collected. After lysing the redblood cells, 0.4×10⁶ of the splenocytes were incubated either withoutany peptide, 10 μg/ml of a non-relevant peptide (IRBP₁₋₂₀, or 10 μg/mlof the MOG₇₉₋₉₆ peptide in 100 μl of complete RPMI medium in each wellof a 96 well plate. After 72 hours, IFN-γ and IL-17 levels in theculture supernatants were measured by respective ELISA followingstandard protocols.

In Vitro Th1 and Th17 Polarization Assays:

CD4⁺T cells were isolated from gender and age-matched WT or CD6 KO miceby negative selection using magnetic beads (EasySep™ Mouse CD4⁺T CellIsolation Kit, STEMCELL Technologies, Canada), then cultured under Th1or Th17 polarization conditions following previously publishedprotocols. Harrington, L. et al. Nature Immunology 6(11): 1123-1132(2005). In brief, CD4⁺T cells (2×10⁴ cells per well) were activated withplate-bound 5 anti-CD28 (BioLegend), then cultured at 37° C. for 5 days.For Th1 polarization, activated cells were cultured in the presence of20 ng/mL recombinant mouse IL-2, 25 ng/mL recombinant mouse IL-12(PeproTech), and 10 μg/mL neutralizing anti-IL-4 antibody (BioLegend).For Th17 differentiation, activated T cells were cultured in thepresence of 20 ng/mL recombinant mouse IL-6, 5 ng/mL recombinant moueTGF-β (PeproTech), and 10 μg/mL neutralizing anti-IL-4 and anti-IFN-γantibodies (BioLegend). On days 1, 2, and 3, cells were stained with 5μL of annexin V per sample followed by flow cytometric analysisaccording to a manufacturer-provided protocol (Annexin V ApoptosisDetection Kit, BD Biosciences). For the BrdU incorporation assay, cellswere cultured in the presence (10 μM), and at different time points, theproliferation of the cells was assessed by measuring BrdU incorporationusing a BrdU ELISA kit (Roche). Finally, differentiated Th1 and Th17cells (day 5) were quantitated by intracellular staining of IFN-γ andIL-17. Harrington, L. et al. Nature Immunology 6(11): 1123-1132 (2005).

T Cell Activation Marker Regulation Analysis:

Purified WT or CD6 KO CD4⁺T cells were activated by 1 μg/ml ofanti-mouse-CD3 and anti-mouse-CD28 (Biolegend, San Diego, Calif.); 5hours later, expression levels of activation markers CD25 and CD69 onthe activated CD4⁺T cells were measured by flow cytometry after stainingwith 2 μg/ml of both PE-anti-mouse CD69 and APC-anti-mouse CD25 mAbs(BioLegend, San Diego, Calif.).

Mouse Primary BMEC Isolation:

Mouse BMEC were isolated following a previously published protocol.Ruck, T. et al. J Vis Exp (93): e52204 (2014). In brief, mouse brainswere isolated, and the brainstems, cerebella, thalami, and meninges wereremoved under a dissecting microscope. The remaining tissue was mincedand digested by 5 mg/mL collagenase CLS2 (Worthington Biochemical) inDMEM for 1 hour at 37° C., then washed with 20% (vol/vol) BSA-DMEM andcentrifuged at 1,000×g for 20 minutes at 4° C. The pellet wasresuspended in 1 mg/mL collagenase/dispase (Worthington Biochemical) andincubated for another 1 hour at 37° C. After the final washing, theresultant cells were cultured in endothelial cell medium (PeproTech).The isolated BMEC purity was determined by flow cytometric analysisafter staining the cells with an anti-CD34 mAb (BioLegend).

Transwell T Cell Migration Assay:

Isolated WT BMEC were cultured onto the upper chambers of 3-μm pore sizeculture inserts (Falcon) in a 24-well transwell plate. After the cellsformed a monolayer, 0.6×10⁶ nylon wool-enriched splenic T cells[activated by 1 μg/mL of both anti-mouse CD3 and anti-mouse CD28 mAbs(BioLegend)] from matched WT and CD6 KO mice were added into the insertswith 20 ng/mL of mouse CCL2 [chemokine (C-C motif) ligand 2] (PeproTech)in the lower chambers (to facilitate T-cell migration), and cultured at37° C. After 18 hours, the number of T cells remaining on top of theBMEC monolayer in the culture inserts and the number of migrated cellsin the bottom chamber were counted by using a hemacytometer after Trypanblue staining and by flow cytometry. The percentages of cells remainingin the upper culture inserts and those that migrated into the lowerchamber were calculated by using the following formula: %=cells in theupper inserts (or cells in the lower chambers)/(cells in the upperinserts+cells in the lower chambers)×100%.

Development of the CD6 Humanized Mice:

CD6 humanized mice were developed by first generating a human CD6 (hCD6)transgenic (Tg) mouse in which hCD6 cDNA expression is driven by a humanCD2 promoter (Zhumabekov et al., Journal of immunological methods 185:133-140 (1995)) following conventional protocols at the transgenic corefacility of Case Western Reserve University. An hCD6 high-expressing Tgmouse was identified by flow cytometry and bred with mCD6 KO mice togenerate CD6 humanized mice (DBA-1 background). The CD6 humanized micewere typed by flow cytometry using respective anti-mCD6 and anti-hCD6mAbs to ensure the presence of hCD6, but the absence of mCD6 on T cells(FIG. 7A, B).

EAE Induction and Treatment Studies:

EAE was induced in the CD6 humanized mice by active immunization withMOG₇₉₋₉₆ peptide as described above. Seven days after immunization, whenmice developed mild clinical signs of EAE, they were randomly separatedinto two groups. One group was injected i.p. with 0.4 mg of a mouseanti-human CD6 IgG (UMCD6) (Singer, N. et al., Immunology 88(4): 537-543(1996)) (0.4 mg per mouse) in the form of diluted ascites, and the othergroup received the same amount of purified mouse IgG (JacksonImmunoResearch) as controls. Mice were then monitored and clinicalscores recorded for another week. At the end of experiments, mice wereeuthanized, and peripheral blood lymphocytes were analyzed forpercentages of CD4⁺, CD8⁺, or CD6⁺T cells, splenocytes were used tocarry out antigen-specific Th1 and Th17 recall assays, and spinal cordswere analyzed by the same histological and histochemical assays asdescribed above.

Statistical analysis: All experiments were repeated at least twice. Todetermine whether groups were statistically different, the clinicalscores were analyzed by the ANOVA test while other results were comparedusing the Student t test. A p value <0.05 was considered significant.

Results and Discussion

To study the role of CD6 in MS, experimental autoimmuneencephalomyelitis (EAE) was induced in wild-type (WT) and CD6-knockout(KO) mice (FIG. 1) (both on DBA/1 background) by subcutaneousimmunization of MOG₇₉₋₉₆ peptide. Abdul-Majid et al., J. Neuroimmunol,111: 23-33 (2000). These experiments demonstrated that CD6 KO mice wereprotected compared to WT mice that developed severe EAE followingimmunization (FIG. 2A). Consistent with markedly reduced EAE severity,CD6 KO mice had reduced T cell infiltration and diminished demyelinationin spinal cords (FIG. 2D, 2E). Recall assays showed decreasedMOG₇₉₋₉₆-specific Th1 and Th17 responses in CD6 KO mice compared withthose from WT mice (FIG. 2B, 2C). These results demonstrate that CD6 isrequired for the development of EAE, potentially by regulatingpathogenic T cell responses and/or T cell infiltration into the CNS.

To elucidate the mechanism by which lack of CD6 reduces pathogenicTh1/Th17 responses and ameliorates disease severity in EAE, CD4⁺T cellswere isolated from naïve WT and CD6 KO mice, then cultured under Th1 orTh17 polarization conditions, followed by flow cytometric analysis ofintracellular IFNγ (Th1) or IL-17(Th17). In these experiments CD6 KOCD4⁺T cells had impaired Th1 and Th17 development compared to WT CD4⁺Tcells (FIG. 3).

Previous studies using anti-CD6 mAbs to study the role of CD6 in T cellactivation generated conflicting results. Some of the data suggest thatCD6 provides co-stimulatory signals to enhance T cell activation andsome suggest that CD6 inhibits T cell activation. Bott, C. et al., IntImmunol 5(7): 783-792 (1993); Singer, N. et al., Immunology 88(4):537-543 (1996). To clarify the role of CD6 in T cell activation, WT andCD6 KO T cells were activated using anti-CD3 and anti-CD28 mAbs for 5hours, then measured upregulation of T cell activation markers CD25 andCD69. The results demonstrate that, compared to WT T cells, CD6 KO Tcells showed augmented upregulation of both CD25 (FIG. 4A) and CD69(FIG. 4B), suggesting that CD6 is a negative regulator of T cellactivation.

The discovery that CD6 is a negative regulator of T cell activationappears to conflict with results from the above EAE studies which showeddecreased Th1/Th17 responses in CD6 KO mice. To address this paradox, WTand CD6 KO CD4⁺T cells were again activated under Th1 or Th17polarization conditions and T cell apoptosis was compared at 5, 24, 48and 72 hours by Annexin V staining. After activation under both Th1 andTh17 polarization conditions, CD6 KO T cells underwent significantlymore apoptosis (Annexin V⁺) than WT T cells (FIG. 5A, 5B).

In addition to activation and survival, proliferation of activated Tcells also governs the outcome of a T cell response. Proliferation ofactivated WT and CD6 KO T cells was therefore measured under Th1 or Th17polarization conditions at 5, 24, 48 and 72 hours after activation, by aBrdU incorporation assay. In the absence of CD6, activated T cells underboth Th1 and Th17 polarization conditions had significantly reducedproliferation (FIG. 5C, 5D).

In EAE, activated pathogenic Th1 and/or Th17 cells need to migratethrough the BBB into the CNS to initiate local inflammation, and BMECsare an important component of the BBB. To determine whether CD6 has aneffect on activated T cell migration through the BBB, BMECs were firstisolated from naïve WT mice following an established protocol (Ruck, T.et al., J Vis Exp (93): e52204 (2014)) (FIG. 6A, B), then these cellswere grown into monolayers on culture inserts in transwell cultureplates. Carboxyfluorescein succinimidyl ester (CFSE)-labeled WT or CD6KO T cells activated with anti-CD3/anti-CD28 mAbs were added onto thetop of the monolayer of BMEC; CCL2 [Chemokine (C-C motif) ligand 2] wasadded to the bottom of the transwells to induce T cell migration. After18 hours of incubation, WT T cells migrated better than the CD6 KO Tcells through the BMEC monolayer (FIG. 6C), suggesting that CD6 on Tcells is required for activated T cells to efficiently migrate throughthe BBB to initiate inflammation in EAE.

To test the potentials of existing mouse anti-human CD6 mAbs for futurehumanization and clinical development to treat MS patients, a human CD6transgenic mouse (hCD6 Tg) was generated in which human CD6 cDNAexpression is driven by a human CD2 promoter/locus control region toreproduce the relative restriction of human CD6 to T-cells. de Boer etal., Eur J Immunol 33: 314-325 (2013). After verifying the expression ofhuman CD6 protein on lymphocytes in the resultant Tg mice, the human CD6Tg mice was backcrossed onto the DBA-1 background, then the human CD6 Tgmice was bred with the mouse CD6 KO mice, and CD6 humanized mice that donot express mouse CD6 (FIG. 7A) but instead express human CD6 in vivo(FIG. 7B) were generated.

To demonstrate that human CD6 can replace mouse CD6 function in vivo andthat these newly developed humanized mice can be used to testCD6-targeted reagents that have potential in treating human diseases,and to evaluate the mouse anti-human CD6 mAb for its potential intreating MS, EAE was induced in the humanized CD6 mice (DBA-1background). Humanized mice were immunized with MOG₇₉₋₉₆ peptide in CFAtogether with pertussis toxin, per protocol described above, then thedevelopment of EAE was assessed by monitoring clinical scores daily.Once mice showed mild signs of EAE clinically, half of the mice wererandomly treated with a mouse anti-human CD6 IgG (UMCD6) (˜0.4 mg/mouse)and the other half with the same amount of mouse IgG by i.p. injection,then the mice were continued to be monitored daily Immunological andhistopathological assays were also carried out as described. Theseexperiments showed that, like WT DBA-1 mice, humanized CD6 micedeveloped severe EAE (FIG. 7C), indicating that transgenic expression ofhuman CD6 can replace the function of mouse CD6 in mice, and supportingthe hypothesis that using CD6 humanized mice can predict the effects ofCD6-related reagents in human MS. Compared with severe EAE thatdeveloped in humanized CD6 mice treated with control mouse IgG, EAEprogression in the treated mice was halted, and these mice showed littleclinical evidence of EAE 7 days after treatment (FIG. 7C). Recall assaysshowed significantly reduced MOG-specific Th1 (FIG. 7D) and Th17responses (FIG. 7E) in the active treatment group compared to control.Further, histopathological assays showed markedly decreased spinal cordinflammation (FIG. 7F) and reduced demyelination (FIG. 7G) in thetreated mice.

Because CD6 is present on all T cells, one possible mechanism by whichthe anti-CD6 mAb ameliorates EAE severity could be depletion of T cells.To test this, CD4⁺ and CD8⁺T cell percentages in the UMCD6-treated andcontrol IgG-treated mice was assessed by staining the peripheralleukocytes with anti-CD4, anti-CD8 and a polyclonal anti-hCD6 IgG (R&D).These experiments found that CD4⁺, CD8⁺ and CD6⁺T cell populations didnot significantly change between the treated and control groups (FIG.8), suggesting that UMCD6 mAb attenuated EAE disease severity in the CD6humanized mice neither by T cell depletion nor by modulating CD6 on Tcells.

These data are the first to provide in vivo evidence demonstrating thatlack of CD6 protects mice from CNS in EAE in association with reducedpathogenic Th1/Th17 responses and decreased T-cell infiltration into theCNS. Additionally, a mouse anti-human CD6 mAb (UMCD6) was shown to behighly effective in treating EAE without depleting T cells.

CD6 was shown to be a negative regulator of T cell activation and, atthe same time, a positive regulator of T cell proliferation andsurvival. The cumulative effects of CD6 on T cell activation,proliferation, and apoptosis together result in CD6 KO T cellsdifferentiating into far fewer IFN-γ-producing Th1 or IL-17-producingTh17 cells compared with WT T cells. The observation that recallresponses to MOG peptide ex vivo showed reduced IFNγ and IL-17 secretionin CD6 KO splenocytes compared to WT splenocytes support that absence ofCD6 reduces production of pro-inflammatory cytokines during memoryresponses.

Even though certain CD6 polymorphisms have been associated withsusceptibility to MS (International Multiple Sclerosis Genetics C., PLoSOne 6(4): e18813 (2011); Heap, G. et al., Hum Mol Genet 19(1): 122-134(2010)), the pathogenic role of CD6 in MS is still unclear.Surprisingly, in in vitro assays, activated T cells from patientscarrying a CD6 risk allele have impaired proliferation comparing to Tcells from donors carrying the non-risk allele. Kofler, D. et al.,Journal of Immunology 187(6): 3286-3291 (2010). By studying WT and CD6KO mice in EAE, the inventor found that the absence of CD6 protectedmice from CNS injury in EAE indicating that CD6 is required for thedevelopment of EAE, and potentially, MS. The inventor's in vitro T cellactivation, proliferation and survival studies provided insights intothe mechanisms underlying the observed reduced MOG-specific Th1 and Th17responses in the CD6 KO mice in EAE. It appears that after EAE inductionin the CD6 KO mice, although MOG-specific T cells were initiallyactivated more robustly, their differentiation into IFNγ-secreting Th1and IL-17-secreting Th17 cells was less efficient and they died fasterthan did T cells in WT mice, leading to reduced MOG-specific Th1/Th17responses, and, eventually, attenuated EAE.

The inventor's H&E studies also showed that there was significantlyreduced cell infiltration in the CNS of CD6 KO mice after EAE induction.To distinguish whether CD6 expression also affected the ability of Tcells to infiltrate to the CNS, the inventor performed in vitro T cellmigration assays comparing the capacity of CD6 KO T cells and WT T cellsto migrate through a monolayer of BMEC. The data showed that CD6 is alsorequired for T cells to infiltrate with optimal efficiency through theBMEC monolayer. This implies that CD6 is important for migration of Tcells through the BBB into the CNS, which is known to be a critical stepin development and/or progression of both EAE and MS.

Speculation that CD6 is a good target for treating autoimmune diseasesincluding MS has existed for decades (Pinto, M & Carmo, A., BioDrug27(3): 191-202 (2013)) but, the first and only clinical study conductedmore than 30 years ago using a T cell-depleting mouse anti-human CD6 IgMfor treating MS patients was inconclusive. Hafler, D. et al., Neurology36(6): 777-784 (1986). Data that CD6 deficiency leads to reduced Th1 andTh17 polarization in vitro and that CD6 KO mice are protected from CNSinjury in EAE in vivo strongly argue that CD6-targeted reagents, usefulfor treating EAE, merit re-evaluation as a potential approach to MS.Since all the available anti-human CD6 mAbs were developed in mice, andprevious studies suggest that CD6 binds to its ligand without speciesrestrictions (Bowen, M., et al., Eur J Immunol 27(6): 1469-1478 (1997)),the inventor developed a CD6 humanized mouse in which human CD6 replacesmouse CD6 on T cells. These animals can be used to screen mouseanti-human CD6 mAbs for future development without confoundingimmunogenicity issues in mice. The inventor's results that CD6 KO miceare resistant to EAE induction and that the restoration of human CD6 inthe CD6 KO mice (CD6 humanized mice) is associated with severe EAE afterimmunization provide clear evidence that human and mouse CD6 functioninterchangeably in mice as previously predicted. Bowen, M. et al., Eur JImmunol 27(6): 1469-1478 (1997). Thus, these CD6 humanized mice areinvaluable to identify effective human CD6-targeted reagents, includinghuman CD6-targeted mAbs in the EAE model of human MS and potentially inother models of human autoimmune diseases.

While there is no current CD6-related clinical trial in the US andEurope, Itolizumab, an anti-human CD6 mAb developed in Cuba has beeneffective in reducing pathogenic T cell responses in psoriasis patientsand was recently approved for treating psoriasis in India. Menon, R &David, B, Clin Cosmet Investig Dermatol 8: 215-222 (2015). Itolizumabcombined with methotrexate has also been reported to reduce T cellnumbers and pro-inflammatory cytokine levels in patients with rheumatoidarthritis, although the clinical outcomes still need to be defined.Aira, L, et al., mAbs 8(1): 187-195 (2016). Surprisingly, Itolizumabbinds to domain 1 of CD6 (Alonso, R, et al., Hybridoma (Larchmt) 27(4):291-301 (2008)) and it does not block the interaction between CD6 andits currently known ligand, CD166, which binds to domain 3 of CD6.Chappell, P, et al., Structure 23(8): 1426-1436 (2015). Interestingly,the anti-human CD6 mAb (UMCD6) that the inventor used to treat EAE inCD6 humanized mice also binds to domain 1 of CD6 and does not blockCD6-CD166 interaction. Singer, N. et al., Immunol Lett 58(1): 9-14(1997). Thus both in vitro and in vivo studies employing anti-CD6 mAbssuggest that the CD6-CD166 interaction might not be critical for CD6function in disease, at least not in psoriasis or MS. Instead, a newligand that interacts with domain 1 of CD6 could be a more important CD6partner than CD166.

A recent report has used CD6^(−/−) mice to assess the role of CD6 in Tcell development and activation. Orta-Mascaró, M., et al., J Exp Med213(8): 1387-1397 (2016). This study found subtle aberrations insingle-positive thymocyte and mature T cell subsets in CD6^(−/−) TCRtransgenic mice. The severity of collagen-induced arthritis was enhancedin CD6^(−/−) mice, in apparent contrast to the current results in theEAE model. It is worth noting that the CIA studies were conducted inC57BL/6 mice, a strain in which the incidence and severity of CIA issubstantially less compared to the DBA-1 strain (the strain that theinventor used for the EAE studies). Additional studies will be requiredto unravel the reasons that underlie the apparent differences betweendistinct autoimmune models and genetically distinct mouse strains in therole of CD6 in the development of autoimmune disease, and whether suchdifferences are paralleled by heterogeneity in the roles of CD6 invarious human autoimmune conditions. Nevertheless, the results in CIAand the current data both highlight an emerging appreciation of thepotentially pivotal role of CD6 in control of T-cell drivenautoimmunity.

In summary, using WT and CD6 KO mice, the inventor demonstrated that CD6is required for the development of EAE. CD6 is a negative regulator of Tcell activation, but a positive regulator of T cell proliferation andsurvival. Therefore, lack of CD6 leads to reduced T cell responses inEAE. In addition, CD6 on T cells is also required for T cellinfiltration through the BBB into the CNS. By developing a CD6 humanizedmice, the inventor showed that human CD6 functions in mice, andidentified UMCD6, a mouse anti-human CD6 mAb, as a potent inhibitor ofEAE. These results encourage exploration of the potential of a humanizedvariant of an anti-CD6 antibody such as UMCD6 to become a newtherapeutic for treating MS and possibly other diseases.

Example 2: Antibody Humanization

This example describes work directed to humanize an anti-CD6 mousemonoclonal antibody without sacrificing the binding affinity. Thehumanization was carried out using two approaches: one is phage displaylibrary-based ‘framework assembly’ method; the other is structure-basedCDR (complementarily determining region) grafting with frameworkback-mutations.

The following experiments were carried out to prepare a humanizedanti-CD5 mouse monoclonal antibody. 1. Generation of phage displaylibrary; 2. Selection of humanized antibodies from phage display libraryand FASEBA screening; 3. Antibody humanization by CDR grafting(structure-based method) with framework back-mutations; and 4.Production and characterization of humanized antibodies.

Methods

Generation of Humanization Phage Display Library:

The combinatorial humanization phage display library was designedaccording to GenScript's proprietary technology ‘Antibody humanizationby framework assembly’; U.S. Pat. No. 9,090,994, the disclosure of whichis incorporated herein by reference. Briefly, frameworks of humanantibodies with high sequence identities to the mouse antibody(GenScript sequencing No. 353920) were selected, randomly combined andassembled. Then the CDRs of mouse antibody were grafted to the assembledcombinatorial human frameworks. The construction of library was carriedout following GenScript's standard operating procedures (SOP).

Isolation of Humanized Antibody Binders from Phage Display Library:

The humanized Fab phage display library was panned against rhCD6-Fc.Individual output phage clones were amplified in 96-deep-well plates andthe amplified phage clones were assayed by ELISA against rhCD6-Fc. Boundphage clones were detected using a HRP/Anti-M13 monoclonal antibody.Phage clones with ELISA signal-to-noise ratios larger than 2.1 wereconsidered to be able to bind rhCD6-Fc. The panning ended after tworounds when a good percentage of phage clones were found to bindrhCD6-Fc. Panning and phage ELISA were carried out following GenScript'sSOP.

Humanization by CDR Grafting: Selection of Acceptor Frameworks:

The variable domain sequences of the parent mouse monoclonal antibodywere searched against the database of germline and rearranged Igvariable region sequences using NCBI Ig-Blast. The variable domainswhich have structures available in Protein Data Bank (PDB) and show thehighest sequence identities to those of parent antibody variable regionsequences were used to generate a homology model of parent antibody.Human sequences with highest identities to parent antibody variableregion sequences were identified. The CDRs of human acceptors werereplaced by those of parent antibody.

FASEBA Screening, Affinity Ranking and Cell Binding Validation by FACS

FASEBA Screening, Affinity Ranking:

DNAs encoding Fab fragments of the output phage were amplified andinserted into pFASEBA vector for screening of the lead antibodies.Individual FASEBA library clones were inoculated and induced forexpression in 96-deep-well plates. ELISA screening was performed toisolate Fabs which recognize rhCD6-Fcspecifically, and the expressionmedium were selected for affinity ranking by BIAcore T200.

BSA was immobilized onto the sensor chip using amine coupling method.The selected Fab-SASA clones secreted to the culture medium wereinjected and captured by BSA via SASA (capture phase). Afterequilibration, antigen rhCD6-Fc was injected for 240 seconds(association phase) followed by the injection of running buffer for 720seconds (dissociation phase). The surface was regenerated before theinjection of another three Fab-SASA clones. Repeat the process until allantibodies are analyzed. Responses of reference flow cell weresubtracted from those of Fab-SASA flow cells during each cycle. Thebinding curves of Fab-SASA clones were aligned at the start ofassociation and normalized at 20 seconds after the stop of antigeninjection using the BIAcore T200 evaluation software in order to displaya more visualized comparison of antibodies. The off-rates of Fab-SASAclones were obtained from fitting the experimental data locally to 1:1binding model. The antibodies were ranked by their dissociation rateconstants. Single cycle kinetics measurement was performed to verify theaffinity ranking result. The selected Fab-SASA clones secreted to theculture medium were injected and captured by BSA via SASA (capturephase). After equilibration, a series of concentrations of antigenrhCD6-Fc (1, 3, 9, 27 and 81 nM) were injected for 500 seconds(association phase) followed by the injection of running buffer for 3000seconds (dissociation phase). The surface was regenerated before anotherinjection of Fab-SASA clones. Responses of reference flow cell andbuffer-only injection were double-subtracted from those of Fab-SASA flowcells during each antigen injection cycle. The binding data were fittedto 1:1 binding model using the BIAcore T200 evaluation software.

BSA was immobilized onto the sensor chip using amine coupling method.The selected Fab-SASA clones secreted to the culture medium wereinjected and captured by BSA via SASA (capture phase). Afterequilibration, antigen rhCD6-Fc was injected for 240 seconds(association phase) followed by the injection of running buffer for 720seconds (dissociation phase). The surface was regenerated before theinjection of another three Fab-SASA clones. The process was repeateduntil all antibodies were analyzed. Responses of reference flow cellwere subtracted from those of Fab-SASA flow cells during each cycle. Thebinding curves of Fab-SASA clones were aligned at the start ofassociation and normalized at 20 seconds after the stop of antigeninjection using the BIAcore T200 evaluation software in order to displaya more visualized comparison of antibodies. The off-rates of Fab-SASAclones were obtained from fitting the experimental data locally to 1:1binding model. The antibodies were ranked by their dissociation rateconstants Single cycle kinetics measurement was performed to verify theaffinity ranking result. The selected Fab-SASA clones secreted to theculture medium were injected and captured by BSA via SASA (capturephase). After equilibration, a series of concentrations of antigenrhCD6-Fc (1, 3, 9, 27 and 81 nM) were injected for 500 seconds(association phase) followed by the injection of running buffer for 3000seconds (dissociation phase). The surface was regenerated before anotherinjection of Fab-SASA clones. Responses of reference flow cell andbuffer-only injection were double-subtracted from those of Fab-SASA flowcells during each antigen injection cycle. The binding data were fittedto 1:1 binding model using the BIAcore T200 evaluation software.

Cell Binding Validation by FACS:

For cell binding validation of Fabon Jurkat cells, flow cytometryanalysis was performed using the culture supernatant. Jurkat cells weregrown to 70-80% confluence and harvested by centrifuge. About 4×10⁵cells per well were washed with PBS twice. 200 μl individual culturesupernatant of protein positive clone was added to the cells andincubated at room temperature for 1 h. After washing with PBS, antibodywas added to the cells for the detection of bound Fab. After 30 minutesincubation, the cells were washed twice with PBS and resuspended in PBS.Cells were analyzed for Fab binding by FACS Calibur (BD Bioscience, SanJose, Calif.) and Flowjo software.

Expression and Purification of Selected Antibodies

Expression of Antibodies:

For each transfection, 125 μg each of light chain and heavy chainexpression plasmids were pre-mixed with 750 μl Polyethylenimine (PEI)stock solution (1 mg/ml) and 10 ml pre-warmed Freestyle 293 medium, thenthe mixture was incubated for 10 minutes at room temperature to allowcomplexes to form. The mixture was added into 240 ml of suspendedHEK293-6E cell culture in Freestyle 293 medium at a cell density ofapproximately 2.0×10⁶ cells per ml. The mixture was transferred into a1-L shaker flask, and incubated at 37° C. and 5% CO₂ on an orbitalshaker rotating with constant shaking at 110 rpm. Pre-warmed TN1 wasadded into the cell culture with a final concentration of 0.5% (w/v) at24h post-transfection. Harvest the conditioned medium on 5-6 dayspost-transfection by centrifugation at 1500×g for 10 minutes to removecells. The supernatants were used for the subsequent antibodypurification.

Characterization of Humanized Antibodies

Affinity Analysis of Antigen-Antibody Interaction:

The affinities of antibodies binding to the antigen rhCD6-Fc weredetermined using a BIAcore T200 SPR system (GE Healthcare) at 25° C. inHBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) Tween20, pH 7.4). For the measurement of three humanized antibody affinitiesfor rhCD6-Fc, rhCD6-Fc (diluted at 2 μg/ml in 10 mM sodium acetate, pH5.0) was immobilized on Series S CMS chips by amine coupling at adensity of ˜100 RU. IgG antibodies were injected through flow cells at aflow rate of 30 μl/min with the concentration ranging from 0.11˜27 nM(three-fold serial dilution). Association time was 700 s followed by3600 s dissociation. At the end of each cycle, sensor surface wasregenerated with 10 mM Glycine-HCl, pH 2.0 (100 μl/min, 12 s for 3times). Responses of reference flow cell and buffer-only injection weredouble-subtracted from rhCD6-Fcflow cell during each antigen injectioncycle. The data of dissociation (kd) and association (ka) rate constantswere obtained by fitting binding data to 1:1 binding model using BiacoreT200 evaluation software. The apparent equilibrium dissociationconstants (KD) were calculated from the ratio of kd over ka.

Binding IC₅₀ Measurement of Humanized IgGs by FACS:

Flow cytometric analysis was performed using the purified antibodies.Jurkat cells were grown to 70-80% confluence and harvested bycentrifuge. About 4×10⁵ cells per well were washed with PBS twice. 200μl of the culture supernatant was added to the cells and incubated atroom temperature for 1 hour. After washing with PBS, antibody was addedto the cells for the detection of bound humanized IgG. After 30 minutesincubation, the cells were washed twice with PBS and resuspended in PBS.Cells were analyzed for IgG binding by using FACS Calibur (BDBioscience, San Jose, Calif.) and Flowjo software. The IC₅₀ was analyzedby GraphPad Prism.

Thermostability Measurement by Circular Dichroism (CD):

The far-UV CD spectra of IgG at increasing temperatures were recordedusing Jasco J-815 CD spectrometer (Jasco Inc.). The temperature-inducedIgG denaturation was recorded by the changes in ellipticity at 202 and208 nm. The temperature at transition midpoint (Tm) was obtained byfitting a two-state denaturation model in which only native anddenatured states are present in the equilibrium to the experimentaldata. Data analysis was carried out using Prism 4 (GraphPad Software,Inc.).

Results

Isolation of humanized antibody binders from phage display library. Tworounds of panning were performed to enrich the humanized binders. Thepositive rate (the number of antigen-specific humanized clone over thenumber of randomly picked clones) of the first round was around 2%.After the second round of panning, the binders were enriched and thepositive rate reached around 40%. The Fab DNAs of the second roundoutput phage were inserted to pFASEBA vector for FASEBA screening.

TABLE 1 Details of panning and phage ELISA experiments. Positive rate ofphage Round Input (pfu) Output (pfu) ELISA 1st 2.0 × 10¹⁰ 3.0 × 10⁴ 1.7%2nd 2.0 × 10¹⁰ 1.5 × 10³ 39.1%

Structure-based humanization by complementarity-determining region (CDR)grafting with framework back-mutations. Homology modeling of the parentantibody variable fragments was carried out using Discovery Studio v5.0(Accelrys). Parent antibody variable region sequences were BLASTsearched against the Protein Database (PDB) antibody database foridentifying the best templates for antibody variable region fragmentsand especially for building the domain interface. Homology models werebuilt using customized Build Homology Models protocol. Disulfide bridgeswere specified and linked. Loops were optimized using DOPE method. Basedon the homology models of parent antibody variable regions, allframework residues in the proximity, i.e. within 5 Å, of all CDRresidues and in the hydrophobic core or V_(H)/V_(L) interface of theantibody were identified. One CDR grafting and seven back-mutationconstructs were designed for this project (CDRs are shown as underlinedamino acids, back-mutation residues are shown in bold, Table 2). Table 2discloses SEQ ID NOS 11, 2, 12, 13, 1, 2, 3, 2, 14, 2, 15, 2, 16, 2, 17,and 2, respectively, in order of appearance.

TABLE 2 Sequences of humanized antibody variants. Name Sequence 1 VHQVQLQESGPGLVKPSETLSLTCTVSGGSISRYSVHWIRQPPGKGLEWIGLIWGGGFTDYNSALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGVAYWGQGTLVTVSS VLDVVMTQSPLSLPVTLGQPASISCKSSQSLLNSDGRTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGPGTKVDIK 3 VHQVQLQESGPGLVKPSETLSLTCTVSGGSISRYSVHWIRQPPGKGLEWIGLIWGGGFTDYNSALKSRVSITVDTSKNQFSLKLSSVTAADTAVYYCAREGVAYWGQGTLVTVSS VLDVVMTQSPLSLPVTLGQPASISCKSSQSLLNSDGRTYLNWFQQRPGQSPKRLIYLVSKLDSGVPDRFSGSGGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGPGTKVDIK 2 VHQVQLQESGPGLVKPSETLSLTCTVSGFSLSRYSVHWVRQPPGKGLEWLGLIWGGGFTDYNSALKSRLTISKDNSKNQVSLKLSSVTAADTAVYYCAREGVAYWGQGTLVTV SS VLDVVMTQSPLSLPVTLGQPASISCKSSQSLLNSDGRTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGPGTKVDIK 4 VHQVQLQESGPGLVKPSETLSLTCTVSGFSISRYSVHWIRQPPGKGLEWIGLIWGGGFTDYNSALKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAREGVAYWGQGTLVTVSS VLDVVMTQSPLSLPVTLGQPASISCKSSQSLLNSDGRTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGPGTKVDIK 5 VHQVQLQESGPGLVKPSETLSLTCTVSGGSLSRYSVHWVRQPPGKGLEWLGLIWGGGFTDYNSALKSRLTISVDTSKNQFSLKLSSVTAADTAVYYCAREGVAYWGQGTLVTVS S VLDVVMTQSPLSLPVTLGQPASISCKSSQSLLNSDGRTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGPGTKVDIK 6 VHQVQLQESGPGLVKPSETLSLTCTVSGFSLSRYSVHWIRQPPGKGLEWIGLIWGGGFTDYNSALKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAREGVAYWGQGTLVTVS S VLDVVMTQSPLSLPVTLGQPASISCKSSQSLLNSDGRTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGPGTKVDIK 7 VHQVQLQESGPGLVKPSETLSLTCTVSGGSISRYSVHWVRQPPGKGLEWLGLIWGGGFTDYNSALKSRLTISVDTSKNQFSLKLSSVTAADTAVYYCAREGVAYWGQGTLVTVSS VLDVVMTQSPLSLPVTLGQPASISCKSSQSLLNSDGRTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGPGTKVDIK 8 VHQVQLQESGPGLVKPSETLSLTCTVSGGSLSRYSVHWVRQPPGKGLEWIGLIWGGGFTDYNSALKSRLTISVDTSKNQVSLKLSSVTAADTAVYYCAREGVAYWGQGTLVTVSS VLDVVMTQSPLSLPVTLGQPASISCKSSQSLLNSDGRTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGGTDFTLKISRVEAEDVGVYYCWQGTHFPFTFGPGTKVDIK

FASEBA Screening, Off-Rate Ranking and Cell Binding Validation by FACS

FASEBA screening, affinity ranking and FACS validation from phagedisplay library. DNAs encoding Fab fragments of second round outputphage were amplified and inserted into pFASEBA vector for screening ofthe lead antibodies by ELISA. 600 clones were selected through FASEBAELISA screening. Forty five clones were sent for DNA sequencing andscreened for binding test against rhCD6-Fc and subsequent off-rateanalysis by the BIAcore T 200. Five clones from the library and Fab2from CDR grafting were selected for single cycle kinetics analysis dueto the low dissociation rate using the BIAcore T200 evaluation software(FIG. 9). And only Fab2 (refer to the sequence of back-mutation 2) with8 point back mutations is comparable to WT Fab (parent chimericantibody) according to single cycle kinetics analysis (FIG. 10).

According to the affinity ranking results, top 10 clones with slowestoff-rate and Fab2 clone from CDR grafting were selected for cell bindingvalidation by FACS. WT Fab (parent chimeric antibody) served as apositive control. FIG. 11 indicates the binding of 11 clones arecomparable with that of WT Fab (parent chimeric antibody) (FIG. 11).

Five back-mutation clones were constructed based on sequence of Fab2 andhomology modeling. Each of DNA sequence encoding the five Fab fragmentswas inserted into pFASEBA vector for screening of the lead antibodies.The five clones along with Fab2 were screened against rhCD6-Fc forbinding test and subsequent off-rate analysis by the BIAcore T200. Threeclones were selected for IgG production and characterization due to thelow dissociation rate using the BIAcore T200 evaluation software (Table3). The nucleotide and amino acid sequences for the selected clones(Fab2, Fab4, and Fab6) are shown in FIGS. 16, 17, and 18, respectively,while the sequences for the V_(L) region are shown in FIG. 19.

TABLE 3 Kinetic data of selected Fab clones. Sample ID K_(d) (1/s) WTFab (parent chimeric antibody)  <5E−06 WT Fab R (parent chimericantibody)  <5E−06 Fab4 1.9E−05 Fab2 2.1E−05 Fab6 3.1E−05 Fab5 R 5.3E−04Fab5 5.6E−04 Fab7 8.9E−04 Fab8 No expression

FACS validation of back-mutation clones binding to Jurkat cells. Fiveback-mutation Fab clones were selected along with Fab2 for cell bindingvalidation by FACS (FIG. 12). According to the FIG. 8, all theback-mutation clones have comparable binding on Jurkat cells exceptFab8, which doesn't express well.

Expression and Purification

The purified Humanized IgG2, Humanized IgG4 and Humanized IgG6 wereanalyzed by SDS-PAGE. The purified proteins have a molecular weight of˜170 kDa with purity of >90% under both reducing and non-reducingconditions (FIG. 13). About 2.0 mg of Humanized IgG2, 2.3 mg ofHumanized IgG4 and 2.6 mg Humanized IgG6 were obtained from each 250 mlculture.

Characterization

Affinity analysis of antigen-antibody interaction. Binding data of eachantibody was processed and fitted to 1:1 binding model using BiacoreT200 evaluation software. All experiment data could be perfectly fittedto the model (FIG. 14). As can be seen from Table 4, all humanizedantibodies have comparable antigen-binding affinities to that of theparent chimeric antibody.

TABLE 4 Kinetics data of rhCD6-Fc chimera/humanized antibodyinteraction. K_(d) (1/Ms) K_(d) (1/s) K_(D) (M) WT (Parent chimericantibody) 6.7E+05 1.2E−05 1.8E−11 Humanized IgG2 (from Fab2) 3.2E+051.3E−05 4.2E−11 Humanized IgG4 (from Fab4) 3.5E+05 1.3E−05 3.8E−11Humanized IgG6 (from Fab6) 6.5E+05 2.1E−05 3.2E−11

The FACS binding assay was also performed for validation of the Jurkatcell/humanized antibody interaction. The binding mean fluorescenceintensity (MFI) & IC₅₀ was listed in Table 5. Three humanized antibodiesshow almost equal Jurkat cell binding activity as parent antibody.

TABLE 5 The binding MFI & IC₅₀ of humanized antibody to Jurkat cell.Samples/ Humanized Humanized Humanized Parent chimeric Conc. (pM) IgG2IgG4 IgG6 IgG 10000 42.7 46.7 45.2 44.4 2000 45 44.4 44.1 42.2 400 31 3735.3 28.8 80 10.9 15 14.8 10.7 16 6.8 7.65 7.9 6.85 3.2 6.18 6.45 6.685.84 0 5.45 5.45 5.45 5.45 IC₅₀ 275.1 185.3 196.9 311.9

Thermostability measurement by Circular Dichroism (CD). For allantibodies, the changes in ellipticity at 202 and 208 nm can be fittedto the two-state denaturation model perfectly (FIG. 15). Thetemperatures at transition midpoint (Tm) obtained by monitoring thechanges in ellipticity at 202 and 208 nm and their average value areshown in Table 6. The Tm values of humanized antibodies are slightlyhigher than that of the parent chimeric antibody, suggesting thathumanization increased its thermostability.

TABLE 6 Temperature at transition midpoint (T_(m)) of parent chimericantibody and humanized antibodies. T_(m) (° C.) 202 nm 208 nm averageParent chimeric antibody 66.05 64.85 65.45 Humanized IgG 2 70.85 69.6570.25 Humanized IgG 4 71.65 70.55 71.1 Humanized IgG 6 70.15 68.95 69.55

CONCLUSION

In this example, anti-CD6 mouse monoclonal antibody was humanized byGenScript. Three humanized antibodies were obtained from structure-basedCDR grafting and framework back-mutations. The humanized antibodies havecomparable affinity to the parent antibody and better thermostability,demonstrating that the result to humanize the mouse antibodies wassuccessful.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material cited herein areincorporated by reference. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood there from. The inventionis not limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

What is claimed is:
 1. A method of treating a T-cell mediated disease ordisorder in a subject by administering to the subject a therapeuticallyeffective amount of an antibody or fragment thereof that specificallybinds to CD6.
 2. The method of claim 1, wherein the disease is anautoimmune disease.
 3. The method of claim 1, wherein the T-cellmediated disease is multiple sclerosis.
 4. The method of claim 1,wherein the antibody is a monoclonal antibody.
 5. The method of claim 1,wherein the subject is human.
 6. The method of claim 5, wherein theantibody is a humanized antibody.
 7. The method of claim 6, wherein thehumanized antibody is selected from the group of antibodies consistingof Fab2, Fab4, and Fab6, or variants thereof including only conservativesequence modifications.
 8. The method of claim 6, wherein the humanizedantibody comprises a heavy chain comprising SEQ ID NO: 1, a light chaincomprising SEQ ID NO: 2, or variants thereof including only conservativesequence modifications.
 9. The method of claim 6, wherein the humanizedantibody comprises a heavy chain comprising SEQ ID NO: 3, a light chaincomprising SEQ ID NO: 4, or variants thereof including only conservativesequence modifications.
 10. The method of claim 6, wherein the humanizedantibody comprises a heavy chain comprising SEQ ID NO: 5, a light chaincomprising SEQ ID NO: 6, or variants thereof including only conservativesequence modifications.
 11. A humanized antibody or antigen-bindingfragment thereof having binding specificity for CD6, wherein theantibody selected from the group of antibodies consisting of Fab2, Fab4,and Fab6, or variants thereof including only conservative sequencemodifications.
 12. The humanized antibody of claim 11, wherein thehumanized antibody comprises a heavy chain comprising SEQ ID NO: 1, alight chain comprising SEQ ID NO: 2, or variants thereof including onlyconservative sequence modifications.
 13. The humanized antibody of claim11, wherein the humanized antibody comprises a heavy chain comprisingSEQ ID NO: 3, a light chain comprising SEQ ID NO: 4, or variants thereofincluding only conservative sequence modifications.
 14. The humanizedantibody of claim 11, wherein the humanized antibody comprises a heavychain comprising SEQ ID NO: 5, a light chain comprising SEQ ID NO: 6, orvariants thereof including only conservative sequence modifications. 15.A kit comprising a humanized antibody or fragment thereof thatspecifically binds to CD6, and a package for holding the antibody. 16.The kit of claim 15, further comprising instructions for using the kitto carry out a method of treating a T-cell mediated disease or disorderin a subject by administering to the subject a therapeutically effectiveamount of the humanized antibody or fragment thereof that specificallybinds to CD6.
 17. The kit of claim 16, wherein the disease is anautoimmune disease.
 18. The kit of claim 16, wherein the T-cell mediateddisease is multiple sclerosis.
 19. The kit of claim 16, wherein theantibody is selected from the group of antibodies consisting of Fab2,Fab4, and Fab6, or variants thereof including only conservative sequencemodifications.
 20. The kit of claim 16, wherein the humanized antibodycomprises a heavy chain comprising SEQ ID NO: 1, a light chaincomprising SEQ ID NO: 2, or variants thereof including only conservativesequence modifications.
 21. The kit of claim 16, wherein the humanizedantibody comprises a heavy chain comprising SEQ ID NO: 3, a light chaincomprising SEQ ID NO: 4, or variants thereof including only conservativesequence modifications.
 22. The kit of claim 16, wherein the humanizedantibody comprises a heavy chain comprising SEQ ID NO: 5, a light chaincomprising SEQ ID NO: 6, or variants thereof including only conservativesequence modifications.